The insulin receptor serves to focus the hormone on particular target tissues as well as to initiate the response of these cells to the hormone. However, it is not known how this signal is transmitted. An understanding of this process is important since many non-insulin dependent diabetics are defective in responding to insulin. Recently it has been demonstrated that the receptor is itself an enzyme, it phosphorylates other proteins on tyrosine residues. This enzymatic activity of the receptor may be important in mediating the responses of cells to the hormone. To further study this process, we have developed monoclonal antibodies that inhibit this enzymatic activity of the receptor. These antibodies will be used to study the role of this activity in insulin action. These antibodies will be microinjected into cells to see if they block various biological effects of insulin. In addition, these antibodies will be utilized to identify proteins in the vicinity of the receptor and thereby identify potential substrates of the receptor kinase. Finally, the antigenic sites recognized by these antibodies will be mapped. After responding to insulin, a cell must terminate its response. One mechanism for accomplishing this is to degrade the hormone. Two enzymes have the ability of degrading the hormone in vitro. One enzyme, called glutathione-insulin transhydrogenase, can be readily purified. Both polyclonal and monoclonal antibodies to this enzyme have been produced and its cDNA has been cloned. However, this enzyme appears to contribute little to the degradation of insulin in the intact cell. Another enzyme which has not been previously purified appears to contribute more to the intracellular degradation of insulin. Recently we have isolated several monoclonal antibodies to this enzyme. These antibodies will be used to examine the structure of this enzyme, its role in insulin degradation and possible interactions with the insulin receptor. In addition, these antibodies will be utilized to isolate the cDNA that encodes the insulin degrading enzyme. An understanding of the enzyme(s) involved in insulin degradation may allow the design of specific inhibitors of the enzyme(s). These inhibitors could be used to treat certain rare patients who have excessive insulin degradation or even to treat patients who have low levels of insulin to make this insulin more active by preventing its degradation.